CN1062312C - Type 1V collagenase-resisting single stranded antibody - Google Patents

Abstract

The present invention relates to a novel anti-type IV collagenase single-chain antibody related to the invasion and metastasis of human cancer cells. The antibody is prepared by the following steps: obtaining mRNA from hybridoma cells secreting anti-IV type collagenase monoclonal antibody, and synthesizing cDNA through reverse transcription. By PCR amplification technology, heavy chain or variable region (V _H ) and light chain variable region (V _L ) genes are obtained, and then anti-IV type collagenase single chain antibody gene is constructed. The gene is cloned into a phage expression vector, transformed into Escherichia coli, expressed to produce a recombinant phage antibody, and the single-chain antibody against type IV collagenase can be obtained through immunoaffinity screening and other steps.

Description

anti-type IV collagenase single chain antibody

The invention belongs to the field of genetic engineering products, and relates to a genetically engineered single-chain antibody for inhibiting tumor cell metastasis.

Invasion and metastasis are important features of malignant tumors and are the main causes of death of tumor patients (Liotta, L.A., Steeg, P.S., Stetler-Stevenson, W.G. 1991). Preventing tumor metastasis is the key to treating tumors. In the process of tumor metastasis, tumor cells first pass through the basement membrane and enter capillaries, and then reach distant sites with blood flow to form secondary tumors. The normal basement membrane is mainly composed of continuous dense collagen and glycoprotein, among which type IV collagen acts as a scaffold in the basement membrane and is a key component to maintain the integrity of the basement membrane structure. When a tumor occurs, the expression and activity of type IV collagenase in the tumor tissue increase significantly, and then degrade the type IV collagen in the basement membrane, destroy the basement membrane, and cause the invasion and metastasis of tumor cells. Therefore look for a kind of material that can inhibit type IV collagenase, and prevent the invasion and metastasis of tumor cells by blocking the activity of type IV collagenase has become an important strategy to control cancer metastasis (Stetler-Stevenson, W, G.1992; Liotta , L, A., et al.1991).

At present, some substances that can inhibit the activity of type IV collagenase have been found at home and abroad, such as metalloproteinase inhibitors, which can combine with type IV collagenase and inhibit the activity of type IV collagenase. Experiments have shown that metalloproteinase inhibitors can reduce the invasiveness of tumor cells. In addition, experiments have shown that treating human melanoma cells with retinoic acid can reduce the mRNA level of collagenase type IV in cells and weaken the invasion ability of melanoma (Sceenath.T., et al.1992). However, the above-mentioned inhibitory effects are all non-specific and may cause side effects in application. So far, no inhibitor that can specifically bind to type IV collagenase has been seen.

The present invention is characterized in that the anti-IV type collagenase antibody is developed by utilizing the high specificity of the antibody. Through the specific combination of antibody and type IV collagenase, the activity of type IV collagenase in tumor tissue is blocked, so as to achieve the purpose of inhibiting tumor cell metastasis.

The invention utilizes bioengineering technology to develop anti-IV type collagenase genetic engineering antibody. The adopted technical scheme is as follows: mRNA is extracted and purified from hybridoma cells secreting type IV collagenase monoclonal antibody. In vitro reverse transcription of cDNA. Using PCR technology, cDNA and a set of antibody light chain or heavy chain variable region primers (Pharmacia), 10×PCR buffer, dNTP final concentration of 2.5mM, and 2 units of Taq DNA polymerase were added to the reaction system. The total reaction volume was 50 μl. After mixing, add mineral oil. Perform 30 cycles of reaction, the conditions of each cycle are: denaturation at 94°C for 30 seconds, annealing at 55°C for 90 seconds, extension at 72°C for 90 seconds, and incubation at 72°C for 10 minutes after the last cycle. The light chain and heavy chain variable region gene amplification products were recovered using Sephagels ^™ Bandprep Kit (Pharmacia). Then, a linker DNA encoding (Gly ₄ Ser) ₃ was used to link the recovered light chain and heavy chain variable region genes under the condition of equimolar concentration through 7 annealing cycles to form a single chain antibody gene. The reaction conditions for each cycle were denaturation at 94°C for 30 seconds and annealing at 64°C for 4 minutes. Using the single-chain antibody gene as a template, in the above reaction system, add a pair of primers containing Sfi 1 at the 5' end and Not 1 restriction site at the 3' end, and carry out 30 PCR cycles, and the condition of each cycle is 94°C Denaturation for 1 minute, annealing at 55°C for 2 minutes, extension at 72°C for 2 minutes. Incubate at 72°C for 10 minutes after the last cycle. PCR products were recovered with Sepheglos ^™ BandPrep Kit. The recovered scFv genes were digested with restriction endonucleases Sfi 1 and Not 1, respectively. Under the action of T4 ligase, it was ligated with the pCANTAB5E vector that had been cut by Sfi1 and Not1. The ligation reaction conditions were overnight at 16°C. The recombinant plasmid containing the single-chain antibody gene was transformed into Escherichia coli TG1 competent cells. With the assistance of the helper phage K07, recombinant phage antibodies were produced. Using the immunoaffinity screening method, the supernatant containing the recombinant phage antibody was co-incubated for 2 hours with type IV gelase coated in a polyethylene plate. Wash the plate 20 times with PBS buffer containing 0.1% Tween 20, 20 ml each time. Discard the PBS, add 10ml of TG1 cells in the logarithmic growth phase to the plate, and incubate at 37°C for 1 hour. Then culture overnight. Centrifuge and take the supernatant. proceed to the next round of screening. After repeating the process of "infection-amplification-screening" 3-4, re-infect TG1 cells, plate and culture overnight. Randomly pick a single colony from the plate and incubate overnight at 30°C. The cells were infected with M13 K07 helper phage and incubated overnight at 37°C. Take the supernatant after centrifugation. Type Ⅳ collagenase phage antibody was screened by ELISA. Those positive clones with the highest antigen binding activity were selected and transformed into Escherichia coli HB2151. Under the induction of IPTG, the soluble type IV collagenase single-chain antibody was expressed. The culture supernatant containing the single-chain antibody was collected and precipitated with 50% ammonium sulfate, and the single-chain antibody was further purified by Sephadex G-75 chromatography column.

The identification of immune activity proves that the single-chain antibody can specifically bind to type Ⅳ collagenase. Antibody COIV-19 is currently the only known small molecule genetically engineered antibody that can specifically bind to type IV collagenase. It will be applied to the treatment of malignant tumors.

Example

1. Cell culture and identification: The hybridoma cells secreting type IV collagenase antibody were cultured in complete RPMI1640 medium containing 15% bovine serum. The incubator contains a gas mixture of 5% _CO2 and a humidity of 98%. The specificity and titer of monoclonal antibody in culture supernatant were identified by ELISA method.

2. Isolation and purification of mRNA: mRNA was extracted and purified from approximately 2×10 ⁷ hybridoma cells with a rapid preparation and purification mRNA kit (Promega).

3 Preparation of cDNA: Using the purified mRNA as a template, synthesize cDNA by reverse transcription.

4. Preparation of antibody variable region genes: use cDNA as a template by PCR method, add a set of light chain or heavy chain variable region primers (Pharmacia) to the PCR reaction system, 5 μl of 10×PCR buffer, and a final dNTP concentration of 2.5 mM, mix well, denature at 100°C for 5 minutes, and add 2 units of Taq DNA polymerase. The total reaction volume was 50 μl. After mixing, add mineral oil. 30 cycles of reaction were performed, and the conditions of each cycle were: denaturation at 94°C for 30s, annealing at 55°C for 90s, extension at 72°C for 90s, and incubation at 72°C for 10 minutes after the last cycle. After the reaction, Sephagles ^™ Bandprep Kit (Pharmacia) was used to recover the amplified products of the light chain and heavy chain variable region genes. DNA sequence analysis proved that the light chain variable region of type IV collagenase antibody is composed of 336 nucleotides, and its gene sequence is as follows:

5 ' TCGGACATCGAGCTCACTCAGTCTCCAACTTCCTTAGCTGCATCTCTGGGGCAGAGGGCCA

CCATCTCATGCAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAA

CCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCT

GGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATC

CTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGGCTTACACGTT

CGGAGGAGGGACCAAGTTGGAAATCAAACGG 3' heavy chain variable region is composed of 348 nucleotides, and its gene sequence is as follows:

5'ATGGCCCAGGTCAAGCTGCAGCAGTCTGGGCCTGACCTGGTGAAGCCCTCTCAGTCACTT

TCACTTACCTGCACTGTCACTGGCTATTCCATTACCGTTCGTTATAGCTGGCACTGGATC

CGGCAGTTTTCCAGGAAACAAACTGGAATGGATGGGCTACATATATTACAATGGTACCACT

AACTACAACCCATCTCTCAGAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAG

TTCTTCCTGAAGTTGAGTTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGA

TGGGATGCTATGGACTATTGGGGCCAAGGCACCACGGTCACCGTCTCC 3’

5. Assembly of the single-chain antibody gene: the recovered light chain and heavy chain variable region genes and the connecting primer (Phamacia) were at equimolar concentrations, 2.5 mM dNTP and 3 units of Taq DNA polymerase were added, and the reaction volume was 50 μl. Seven cycles of annealing were performed after blocking with liquid paraffin oil. The reaction conditions for each cycle were denaturation at 94°C for 30 s and annealing at 64°C for 4 minutes.

6 Amplification of the single-chain antibody gene: using the single-chain antibody gene as a template, in the above reaction system, add a pair of primers containing Sfi 1 at the 5' end and Not 1 restriction site at the 3' end, and perform 30 times of PCR Cycle, each cycle condition is denaturation at 94°C for 1 minute, annealing at 55°C for 2 minutes, and extension at 72°C for 2 minutes. Incubate at 72°C for 10 minutes after the last cycle. PCR products were recovered with Sephgles ^™ BandPrep Kit. DNA sequence analysis proved that the type IV collagenase single-chain antibody gene is composed of 729 nucleotides, and its nucleotide sequence is as follows:

1 ATGGCCCAGGTCAAGCTGCAGCAGTCTGGGCCTGACCTGGTGAAGCCCTTCCAGTCACTT

61 TCACTTACCTGCACTGTCACTGGCTATTCCATTACCGTTCGTTATAGCTGGCACTGGATC

121 CGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATATATTACAATGGTACCACT

181 AACTACAACCCATCTCTCAGAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAG

241 TTCTTCCTGAAGTTGAGTTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGA

301 TGGGATGCTATGGACTATTGGGGCCAAGGCACCACGGTCACCGTCTCCTCAGGTGGAGGC

361 GGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCGAGCTCACTCAGTCTCCA

421 ACTTCCTTAGCTGCATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCAAAAGT

481 GTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCC

541 AGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGC

601 AGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCA

661 ACCTATTACTGTCAGCACATTAGGGAGGCTTACACGTTCGGAGGAGGGACCAAGTTGGAA

721 ATCAAACGG

7 Connection of single-chain antibody gene and expression vector: the recovered single-chain antibody gene was digested with restriction endonucleases Sfi 1 and Not 1 respectively. Then, under the action of T4 ligase, it was ligated with the pCANTAB5E vector that had been cut by Sfi1 and Not1. The ligation reaction conditions were overnight at 16°C.

8. Expression of the single-chain antibody gene: Transform the recombinant plasmid containing the single-chain antibody gene into E. coli TG1 competent cells, spread the transformed cells on LB culture plates containing 100 μg/ml ampicillin, and culture overnight at 37°C.

Collect the transformed bacteria grown on the LB culture plate, dilute the cell suspension with an appropriate amount of SOC medium (containing 100ug/ml ampicillin and 2% w/v glucose) until the A ₆₀₀ light absorption value is about 0.2, and store at 37°C Cultivate until the A ₆₀₀ absorbance value is about 0.4. Add 2.5×10 ⁹ pfu M13K07 helper phage and incubate at 37°C for about 1 hour. After centrifugation, the cell pellet was resuspended in 10 ml of SOC medium containing no glucose, 100 μg/ml ampicillin and 50 μg/ml kanamycin, and cultured overnight at 37°C. The supernatant containing the recombinant phage was collected by centrifugation.

9. Infection-amplification-screening: dilute type IV gluease with 0.05M NaHCO ₃ , pH 9.6 to make the final concentration 10 μg/ml, coat on polyethylene plate, overnight at 4°C. The next day, wash the plate three times with PBS buffer, then add 20ml of 1% BSA blocking solution, and incubate at room temperature for 1h. The plates were then washed three times with PBS. Mix the blocking solution and the supernatant containing the recombinant phage antibody in an equal volume of 1:1, take 20ml of the mixed solution and add it to a plate, and incubate at 37°C for 2 hours. Wash the plate 20 times with PBS buffer solution, each containing 0.1% Tween 20 in 20ml. Discard PBS, add 10ml of TG1 cells in logarithmic growth phase, and incubate at 37°C for 1 hour. Then culture overnight. Centrifuge and take the supernatant for the next round of screening. After repeating the "infection-amplification-screening" process 3-4, re-infect TG1 cells and plate. On the next day, a single colony was randomly selected from the plate, inoculated in 100 μl of SOC culture solution containing 100 μg/ml ampicillin and 2% glucose, and cultured at 30° C. overnight. The transformed cells were then infected with M13 K07 helper phage, cultured at 37°C for 1 hour, centrifuged, and the cell pellets were reselected in culture medium containing 100 μg/ml ampicillin and 50 μg/ml kanamycin, and cultured overnight at 37°C. Take the supernatant after centrifugation.

10 Screen type IV collagenase phage antibody: Coat 96-well enzyme-linked plate with 10 μg/ml type IV collagenase (solution: 0.05M Na ₂ HCO ₃ , pH 9.6), and store at 4°C overnight. The next day, the enzyme-linked plate was blocked with 1% BSA. Incubate for 1 h at room temperature. The plate was then washed three times with PBS. The blocking solution and the supernatant containing the recombinant phage antibody were mixed in an equal volume of 1:1, and 100 μl of the mixed solution was added to each well, and incubated at 37° C. for 2 hours. Wash the plate 4 times, each time 200μl containing 0.1% Tween 20. PBS was discarded, and 100 μl of anti-M13 phage antibody (Phamacia) diluted 1:2000 was added to each well. React at 37°C for 1 hour. After washing 4 times, add substrate hydrogen peroxide and o-phenylenediamine (H ₂ O ₂ -OPD), react at room temperature for 15 minutes, add 50 μl of 2 M H ₂ SO ₄ to terminate the reaction solution, and detect the light absorption value of each well at A ₄₉₅ . One of the positive clones with the highest binding activity to type Ⅳ collagenase was named COIV-19.

11 Preparation of single-chain antibody: transfer the recombinant plasmid containing COIV-19 gene into HB2151 cells. Plates were plated on SOBAG medium and incubated overnight at 30°C. Randomly pick a single colony from the plate, and culture them in 2ml 2xYT medium containing 100μg/ml ampicillin and 2% glucose, and culture overnight at 30°C. After centrifugation, the cells were resuspended in 2 x YT medium containing 1mM IPTG and 100μg/ml ampicillin, and cultured at 30°C for 20 hours. Centrifuge and collect the supernatant. The supernatant was precipitated with 50% ammonium sulfate, purified by Sephadex G-75 chromatography column, and finally soluble anti-type IV collagenase single chain antibody was obtained with a molecular weight of 29KD. The type IV collagenase single-chain antibody deduced from the DNA sequence is composed of 243 amino acids, and its sequence is:

MAQVKLQQSGPDLVKPSQSLSLTCTVTGYSITVRYSWHWIRQFPGNKL

EWMGYIYYNGTTNYNPSLRSRISITRDTSKNQSSLKLSSVTTEDTATYY

CARWDAMDYWGQGTTWTVSSGGGGSGGGGSGGGGSDIELTQSPTSLA

ASLGQRATISCRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLES

GVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIREAYTFGGGTKLEIKR

Claims (5)

1. An anti-type IV collagenase single-chain antibody gene is characterized in that it consists of the following 729 nucleotides: 1 ATGGCCCAGGTCAAGCTGCAGCAGTCTGGGCCTGACCTGGTGAAGCCCTTCAGTCACTT 61 TCACTTACCTGCACTGTCACTGGCTATTCCATTACCGTTCGTTATAGCTGGCACTGGATC 121 CGGCAGTTTCCAGGAAACAAACTGGAATGGATGGGCTACATATATTACAATGGTACCACT 181 AACTACAACCCATCTCTCAGAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAG 241 TTCTTCCTGAAGTTGAGTTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGA 301 TGGGATGCTATGGACTATTGGGGCCAAGGCACCACGGTCACCGTCTCCTCAGGTGGAGGC 361 GGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGACATCGAGCTCACTCAGTCTCCA 421 ACTTCCTTAGCTGCATCTCTGGGGCAGAGGGCCACCATCTCATGCAGGGCCAGCAAAAGT 481 GTCAGTACATCT6GCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCC 541 AGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGC 601 AGTG6GTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCA 661 ACCTATTACTGTCAGCAGATTAGGGAGGCTTACACGTTCGGAGGAGGGACCAAGTTGGAA 721 ATCAAACGG 2. The single-chain antibody gene according to claim 1, characterized in that the single-chain antibody gene contains a heavy chain variable region gene (V _H ), which consists of the following 348 nucleotides: 5'ATGGCCCAGGTCAAGCTGCAGCAGTCTGGGCCTGACCTGGTGAAGCCCTCTCAGTCACTT TCACTTACCTGCACTGTCACTGGCTATTCCATTACCGTTCGTTATAGCTGGCACTGGATC CGGCAGTTTTCCAGGAAACAAACTGGAATGGATGGGCTACATATATTACAATGGTACCACT AACTACAACCCATCTCTCAGAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAG TTCTTCCTGAAGTTGAGTTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGA TGGGATGCTATGGACTATTGGGGCCAAGGCACCACGGTCACCGTCTCC 3’ 3. The single-chain antibody gene according to claim 1, characterized in that the single-chain antibody gene contains a light chain variable region gene (V _L ), which consists of the following 336 nucleotides: 5'TCGGACATCGAGCTCACTCAGTCTCCAACTTCCTTAGCTGCATCTCTGGGGCAGAGGGCCA CCATCTCATGCAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAA CCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCT GGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATC CTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGGCTTACACGTT CGGAGGAGGGACCAAGTTGGAAATCAAACGG 3' 4. The single-chain antibody encoded by the single-chain antibody gene of claim 1 is characterized in that it is composed of the following 243 amino acids: MAQVKLQQSGPDLVKPSQSLSLTCTVTGYSITVRYSWHWIRQFPGNKL EWMGYIYYNGTTNYNPSLRSRISITRDTSKNQSSLKLSSVTTEDTATYY CARWDAMDYWGQGTTWTVSSGGGGSGGGGSGGGGSDIELTQSPTSLA ASLGQRATISCRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLES GVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIREAYTFGGGTKLEIKR 5. The application of the single-chain antibody according to claim 4 in the preparation of drugs for diagnosis, treatment or prevention of tumors.